PROJECT SUMMARY The pharyngeal pouches develop into the thymus, thyroid and parathyroid glands and contribute to formation of the ear and tonsils. They also provide signals that are essential to the morphogenesis of the craniofacial skeleton. In this latter role, the specific morphology and position of the pouches relative to the mesenchyme of the pharyngeal arches is critical to instruct skeletal formation. Despite these essential roles for proper development of the vertebrate head, pharyngeal pouch development in mammals remains poorly understood. Pharyngeal pouch formation consists of two separate morphogenetic processes, lateral out-pocketing and proximal-distal extension. The work outlined in this proposal focuses on understanding the role of Fgf8 in the latter process. Using an allelic series of Fgf8 mutant mice, embryos of different Fgf8 dosages can be generated, including both a mild and severe mutant. In contrast to zebrafish, in both mouse Fgf8 mutant genotypes, the first pharyngeal pouch (pp1) out-pockets to contact the ectoderm but fails to extend along the proximal-distal axis. In mild mutants, pp1 is reduced in size, but does extend. In the severe mutant, pp1 does not extend proximo-distally and the ectodermal cleft is also hypoplastic and disorganized, contributing to failure of the first and second arches to separate distally. Severe mutants have small, round pouches in which cells appear to stack upon each other. Together, these data suggest that Fgf8 has additional roles in pharyngeal pouch formation beyond directing lateral out-pocketing, which may include proximal-distal extension of both the pharyngeal endoderm and ectoderm. Previous research has suggested that Fgf8 regulates cell polarity, and that polarity of the actin cytoskeleton is essential to pharyngeal pouch extension. The specific hypothesis to be tested by the proposed research is that Fgf8 has a paracrine function regulating proliferation and polarity of pharyngeal pouch epithelial cells. This hypothesis will be tested through two specific aims. In Specific Aim 1, proliferation and polarity in pouch epithelial cells will be quantified in embryos in which Fgf8 has been reduced globally. In Specific Aim 2, pouch shape will be evaluated in embryos in which Fgf8 is specifically ablated in the pharyngeal mesoderm or ectoderm. The future research goals of this work are to investigate genetic and developmental interactions underlying variation in craniofacial morphogenesis, particularly variation in the severity and penetrance of craniofacial disorders. The Fgf8 allelic series exhibits a large range of morphological variation, including the bilateral variation present in both mutant genotypes. Both Fgf8 mutant genotypes exhibit directional asymmetry of the jaw, exemplified by Fgf8Neo/Neo mice in which unilateral fusion of the jaw on the left side only is observed in 33% of neonates. Although it has yet to be shown directly, directional asymmetry is likely due bilateral asymmetry in Fgf8 expression in the cranial mesoderm as a consequence of heart development. Facial asymmetry and heart defects are associated in several syndromes, notably CHARGE and DiGeorge (22q11 deletion) syndrome, which have similar phenotypes to Fgf8 mutants.